Aminopterin, or 4-amino-pteroyl-L-glutamic acid, is a potent antifolate [see Franklin, U.S. Pat. No. 2,575,168]. Aminopterin binds and inhibits dihydrofolate reductase (DHFR) from a variety of species, including humans. Synthesized in 1946 by the Lederle Laboratories, a division of American Cyanamid Co., aminopterin was marketed in 0.5 mg tablets in 1953 for the treatment of childhood leukemia. In 1965, the marketing of aminopterin as a pharmaceutical in the United States ceased.
In 1951, Gubner treated patients with rheumatoid arthritis, psoriatic arthritis, uncomplicated psoriasis, and atopic dermatitis with 0.75-2 mg/day for durations of approximately 1 week and greater, or cumulative weekly doses of greater than 5.25 mg [Gubner et al., Am J. Med. Sci. 22:176, 1951; and Gubner, Arch. Derm., Chicago 64:688, 1951]. Although some patients improved, the majority of patients developed toxic reactions including stomatitis, nausea, diarrhea, and alopecia that necessitated discontinuation of the drug. Gubner concluded that “the toxic effects of aminopterin place practical limitations on its use as a therapeutic agent” [Gubner et al., Am. J. Med. Sci. 22:176, 1951].
In 1955, Rees et al. treated 171 patients with psoriasis with five dosage schedules using the 0.5 mg tablet that comprised: (i) 1 tablet daily for six days; (ii) 1 tablet daily for six days, one week rest, then 1 tablet daily for six days; (iii) 1 tablet daily for 12 days; (iv) 2 tablets daily for six days; and (v) 2 tablets daily for three days, then 1 tablet daily for six days [Rees et al., AMA Arch. Derm. 72(2):133-43, August 1955]. A rapid clearing of psoriatic lesions were noted, with toxic reactions occurring with a frequency of 0%, 2.5%, 13%, and 30% on schedules (i), (ii), (iii) and (iv), respectively. Too few patients were treated with schedule (v) to assess. Toxic reactions included stomatitis, alopecia, and leucopenia. When the above schedules were concluded, the psoriatic lesions invariably recurred, usually within weeks. Some patients were given multiple courses of the above schedules, with rest periods between courses.
In 1958, Edmundson and Guy treated patients with a schedule comprising 1 tablet daily for six days, withdrawal for three days, and again daily for six days, for a total of 6 mg in 12 doses [Edmundson and Guy, AMA Arch. Derm. 78(2):200-3, August 1958]. Improvement was noted, and remissions typically lasted several months.
In 1959, Rees and Bennett report the treatment of 329 patients with psoriasis using the same schedules from their 1955 study [Rees and Bennett, J. Invest. Dermatol. 32(1):61-66, January 1959). Courses of the schedules were repeated in some patients every 3 weeks to once every 3 years, although it is not disclosed which schedules were repeated. The overall incidence of toxicity was 21% and the most common toxic reactions were stomatitis and intensification of the lesions, followed by alopecia, GI disturbances, and leucopenia. The authors noted that treatment should be discontinued at the slightest hint of a toxic reaction.
In 1961, Rees and Bennett compared the effect of daily doses of 0.5 mg aminopterin against daily doses of 2.5 mg methotrexate using the same schedules from their 1955 study [Rees and Bennett, Arch. Dermatol. 83:970-72, June 1961]. In 1963, Strakosh also compared the effect of daily doses of 0.5 mg aminopterin against daily doses of 2.5 mg methotrexate according to schedules comprising: (i) 1 tablet daily for 12 days, followed by one-week's rest, and repeated as often as deemed advisable; (ii) 1 tablet daily for 3 days followed by three-days' rest period and repeated until 12 tablets in all were given, and then followed by one-week's rest period and repeated as often as deemed advisable (i.e., four cycles interrupted by a week rest each); and (iii) any variation of (i) and (ii) above [Strakosch, Dermatologica 126:259-267, 1963]. Both concluded that methotrexate is less toxic and less efficacious than aminopterin in treating psoriasis. However, the doses compared were not equipotent to one another, with methotrexate being used in an amount 4-fold less than would be required to be equipotent with aminopterin. Thus, by modern standards no conclusions could be drawn regarding the actual relative efficacy, toxicity, or therapeutic index of these antifolates. Later, Rees et al. suggest the opposite, that methotrexate may be more safe and efficacious than aminopterin in treating psoriasis [Rees et al., Arch. Dermatol. 90:544-52, December 1964].
In 1964, Rees et al. review the literature on the standard of practice with aminopterin in treating psoriasis, describing all known schedules of administration [Rees et al., Arch. Dermatol. 90:544-52, December 1964]. In all cases, dosing schedules were similar, being comprised of daily dosings of tablets for periods greater than 1 week until efficacy or toxicity was observed, at which time dosing was interrupted by rest periods of varying duration.
The prior art are also contains several reports of using aminopterin to reduce inflammation in animal models. In 1952, Gubner et al. demonstrated the efficacy of cumulative weekly doses of 0.3 mg/kg aminopterin in the rat formaldehyde arthritis model [Gubner et al., J. Invest. Dermatol. 19(4):297-305, October 1952]. In 1964, Page demonstrated the efficacy of cumulative weekly doses of 0.35 mg/kg aminopterin in the rabbit dermal inflammation model, but in the process the animals became severely leucopenic and one subject died [Page, Ann. N.Y. Acad. Sci. 116:950-63, Aug. 27, 1964]. In 1986, Galivan et al. demonstrated the efficacy of cumulative weekly doses of 0.12 mg/kg aminopterin in the rat adjuvant arthritis model, but the animals suffered from severe toxicity [Galivan, et al., Methotrexate in adjuvant arthritis, in Chemistry and Biology of Pteridines 1986. Pteridines and Folic Acid Derivatives, B. A. Cooper and V. M. Whitehead, Editors. 1986, Walter de Gruyter & Co.: Berlin. p. 847-49]. Similarly, in 2000 Andersson et al. demonstrated the efficacy of cumulative weekly doses of >1.5 mg/kg aminopterin in the rat antigen-induced arthritis model, but again the animals suffered from severe toxicity [Andersson, et al., Eur. J. Pharm. Sci. 9(4):333-43, 2000].
Inflammatory diseases, whether of a chronic or acute nature, represent a substantial problem in the healthcare industry. Chronic inflammation is considered to be inflammation of a prolonged duration (weeks or months) in which active inflammation, tissue destruction and attempts at healing are proceeding simultaneously (Cotran, R. S., Kumar, V., and Robbins, S. L., Robbins Pathological Basis of Disease. W.B. Saunders Co., p. 75, 1989). Although chronic inflammation can follow an acute inflammatory episode, it can also begin as an insidious process that progresses with time, for example, as a result of a persistant infection (e.g., tuberculosis, syphilis, fungal infection) which causes a delayed hypersensitivity reaction, prolonged exposure to endogenous (e.g., elevated plasma lipids) or exogenous (e.g., silica, asbestos, cigarette tar, surgical sutures) toxins, or autoimmune reactions against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis). Chronic inflammatory diseases include many common medical conditions such as rheumatoid arthritis, restenosis, psoriasis, multiple sclerosis, surgical adhesions, tuberculosis, chronic inflammatory lung diseases (e.g. asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps and pulmonary fibrosis), periodontal disease (i.e. periodontitis) and polycystic kidney disease.
Multiple sclerosis (MS), affecting 350,000 people (women:men=2:1) in the United States with 8,000 new cases reported each year, is the most common chronic inflammatory disease involving the nervous system. The predominant pathologic findings are demyelination accompanied by disruption of underlying axons (Trapp et al., New Engl. J. Med. 338:278-85, 1998; Prineas, J. W., “Pathology of Multiple Sclerosis” in: Cook, S. D., ed. Handbook of Multiple Sclerosis. New York: Marcel Dekker, Inc., 1990:187-215). The disease affects young adults who usually present with a relapsing, remitting pattern of neurologic involvement and progress to a chronic phase with increasing difficulty in ambulation and coordination. Roughly half of MS cases progress to a more chronic phase. Although the disease does not result in early death or impairment of cognitive functions, it cripples the patient by disturbing visual acuity; simulating double vision; disturbing motor functions affecting walking and use of the hands; producing bowel and bladder incontinence; spasticity; and sensory deficits (touch, pain and temperature sensitivity).
MS is a demyelinating disease of the CNS with unknown cause and no known cure. Though single episodes of demyelination can occur, once the disease is established in multiple sites in the brain, spinal cord, and optic nerves, MS frequently follows a clinically relapsing-remitting course while lesions in the CNS continue to progress. During this phase, an immune mediated inflammatory response to myelin antigens is thought to play a major role in the pathogenesis of developing lesions. Then, in a clinically progressive phase, at least fifty-five percent of patients worsen, with clinical relapses sometimes punctuating their clinical decline. The mechanism of tissue damage to the CNS is not known with certainty in the progressive phase of MS. It is thought, however, that axonal damage, perhaps through some type of immune mediation, is important in this phase of the disease, though some axonal damage certainly occurs during the inflammatory phase. Perivascular infiltration of T lymphocytes and macrophages in brain lesions is one of the characteristics of MS. Activation of myelin-reactive T cells in the periphery is an early event in the MS process. These activated T cells facilitate the production of B cells of antibodies against myelin, and activate macrophages to attack oligodendrocytes in the CNS. Treatment options for patients with MS are limited. Currently, the primary drugs used to treat MS are interferons and glatiramer acetate. Such treatments have been shown to slow, but not arrest, the clinical course of progression in progressive MS.
Experimental Autoimmune Encephlomyelitis (EAS) is an experimental model for human MS (Mackay, et al., Clin. Exp. Immunol. (1973) 15:471-82). The use of this model can be demonstrated in guinea pigs (Lisak, et al., J. Immunol. (1970) 104(6):1435-47); hens (Blaszczyk, et al., Folia biologica (Praha) (1977) 23:299; female rats (Levine, and Sowinski, Arch. Int. Pharmacodyn. (1977) 230:309-318; Badger, et al., Agents and Actions (1989) 27 (3/4):335-7; Desai, and Burton, Agents and Actions (1989) 27 (3/4): 351-355; Vogel, et al., Can. Res. (1969) 29:2249-2253; Przuntek, et al., Neuropharma. (1987) 26(2/3):255-260); male rats (Rosenthale, et al., Arch Int. Pharmacodyn. (1969) 179(2):251-275); rabbits (Brandiss, et al., Ann. NY Acad. Sci. 356-368); and male and female rats (Levy, and Whitehouse, Agents and Actions (1974) (4/2):113-116; Martel, et al., Can. J. Physiol. Pharmacol. (1977) 55:48-51).
Chronic inflammatory lung disease, including for example, asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps, and pulmonary fibrosis, affect many people worldwide. Typically such diseases are characterized by an invasive inflammatory process, and thickening of the affected tissues.
Asthma is an inflammatory disease that is associated with widespread but variable airflow obstruction. Asthma is defined as a “disease characterized by an increased responsiveness of the trachea and bronchi to various stimuli, and manifested by widespread narrowing of the airways that changes in severity either spontaneously or as a result of treatment” (American Thoracic Society). About 5% of the population suffers from asthma. The pathogenesis of asthma is poorly understood. Multiple complex immune system mechanisms probably are involved. Numerous cytokines derived from tissue mast cells, eosinophils, T lymphocytes, macrophages, neutrophils, and other lung cells are critical in initiating and perpetuating the asthmatic response and play important roles in the immunopathogenesis of airways in inflammation (Current Medical Diagnosis & Treatment, 36th ed.). In persons with asthma, the airways narrow (bronchoconstriction) in response to stimuli that don't affect the airways of normal lungs. The narrowing can be triggered by many stimuli (an allergen), such as pollens, dust mites, animal dander, smoke, cold air, and exercise. Bronchodilator drugs that relieve attacks of asthma through non-specific blockade of beta-adrenergenic receptors cause side effects such as rapid heartbeat, restlessness, headache, and muscle tremors. Theophylline is another bronchodilator drug whose administeration must be closely monitored by a doctor, because too little drug in the blood may give little benefit, and too much drug may cause life-threatening abnormal heart rhythms or seizures and patients may also experience insomnia, agitation, and vomiting. Corticosteriods drugs are exceptionally effective at reducing asthma symptoms by blocking the body's inflammatory response but long term use may result in poor wound healing, loss of calcium from the bones, stomach bleeding, premature cataracts, elevated blood sugar levels, hunger, weight gain, mental problems, and stunted growth in children.
The benefits of asthma treatments have been investigated in animals (Nagao, et al., J. Pharma. Pharmacol. (2004), 56:187-196) and humans (Comet, et al., Resp. Med. (2006), 100:411-19; Rabe, et al., Lancet. 368:744-53; Busse, et al., Ann. All. Asth. Immunol. 96:60-68; Johnston, et al., New Engl. J. Med. 354 (15):1589-1600; Bateman, et al., Ann. All. Asth. Immunol 96:679-686).
Systemic lupus erythematosus (SLE) is a disorder of the immune system. Lupus can affect many parts of the body, including the joints, skin, kidneys, heart, lungs, blood vessels and brain. Although patients with the disease may have many different symptoms, some of the most common ones include extreme fatigue, painful or swollen joints (arthritis), unexplained fever, skin rashes, and kidney problems. Other symptoms include chest pain upon deep breathing, unusual loss of hair, pale or purple fingers from cold or stress (Raynaud's phenomenon), sensitivity to sun, swelling (edema) in legs or around eyes, mouth ulcers and swollen glands. New symptoms may continue to appear years after the initial diagnosis, and different symptoms can occur at different times. Several systems of the body may be affected including the kidneys, lungs, CNS, blood vessels, blood and heart.
Several methods of treatment are currently employed to treat SLE. Nonsteroidal anti-inflammatory drugs (NSAIDS) are often used, either alone or in combination, including ibuprofen and naproxen. Common side effects include stomach upset, heartburn, diarrhea and fluid retention. Antimalarials, such as hydrochloroquinone, are also commonly used to treat lupus. They are generally used to treat fatigue, joint pain, skin rashes, and inflammation of the lungs. Side effects include stomach upset, and, rarely, damage to the retina of the eye. Corticosteroids are also used to treat lupus. Examples include prednisone, hydrocortisone, methylprednisone and dexamethasone. Short-term side effects include swelling, increased appetite and weight gain. These side effects generally stop when the drug is stopped. Long-term side effects can include stretch marks of the skin, weakened or damaged bones (osteoporosis and osteonecrosis), high blood pressure, damage to the arteries, diabetes, infections and cataracts. Typically, the higher the dose and the longer they are taken, the greater the risk and severity of side effects. For some patients whose kidneys or CNS are affected by lupus, immunosuppressives may be used. These include cyclophosamide and mycophenolate mofetil. Side effects may include nausea, vomiting, hair loss, bladder problems, decreased fertility, and increased risk of cancer and infection. The risk for side effects increases with the length of treatment.
Polymyositis is a disease of muscle featuring inflammation of the muscle fibers. The muscles typically affected are those closest to the trunk or torso. Polymyositis can be associated with skin rash and is then referred to as dermatomyositis. It also can affect other areas of the body. Weakness of muscles is the most common symptom of polymyositis. The onset can be gradual or rapid. This results in varying degrees of loss of muscle power and atrophy. Patients can also feel fatigue, a general feeling of discomfort and have weight loss and/or low grade fever. Heart and lung involvement can lead to irregular heart rhythm, heart failure, and shortness of breath. Initially, polymyositis is treated with high doses of corticosteroids and are usually required for years. Corticosteroids have many predictable and unpredictable side effects. In high doses they commonly cause increase in appetite and weight, puffiness of the face and easy bruising, sweats, facial hair growth, upset stomach, sensitive emotions, leg swelling, acne, cataracts, osteoporosis, high blood pressure, worsening of diabetes, increased risk of infection, and rarely, avascular necrosis. Further, abruptly stopping corticosteroids can cause flares of the disease and result in other side effects including nausea, vomiting and decreased blood pressure. Immunosupressives may used in the cases where corticosteroids do not adequately improve polymyositis. These include the use of methotrexate and azathioprine. These medications can cause liver and bone marrow side effects and require blood monitoring. Cyclophosphamide, chlorambucil and cyclosporine may also be used, however these compounds also have serious side-effects.
Graft-versus-host-disease (GVHD), an immune effect, is the most common complication among patients who survive beyond 100 days after allogenic hematopoietic cell transplantation. The incidences of acute GVHD (aGVHD) and chronic GVHD (cGVHD) are 30-80% and 30-80%, respectively. GVHD itself and treatment-related infections are the principal causes of nonrelapse mortality. For aGVHD after methotrexate alone or methotrexate in combination with cyclosporine A as prophylaxis regimens, methylprednisone is the best initial therapy. Prednisone and cyclosporine A are considered as the first-line therapy for patients with cGVHD. Other therapeutic options for aGVHD or cGVHD are anti-thymocyte globulin, mycophenolate mofetil, tacrolimus, azathioprine, thalidomide, monoclonal antibodies directly against CD3, CD25, CD52, cytotoxic T-lymphocyte antigen (CTLA)-4 or tumor necrosis factor alpha, extracorporeal photochemotherapy and PUVA therapy. Most immunosuppressive agents require continuous administration of the drug to be effective. Consequently, infectious complications are frequently observed and tremendously increase the treatment cost. Although treatment with cyclosporine and gluccocorticoids can control manifestations of cGVHD in some patients, the therapeutic options are limited for those with steroid-refractory cGVHD.